Epigenetic modifications are heritable changes in gene expression and phenotype without changing in the DNA sequence. Previous studies have shown that epigenetic regulation is involved in many life processes and plays an important role in fat deposition. Moreover, fat deposition is an important factor affecting the meat quality of livestock and poultry. Studying the mechanism of fat deposition is a significant aspect of breeding. In this review, the research progress of DNA methylation, mRNA modification, histone modification, chromosomal remodeling, and non-coding RNA regulation on the mechanism of fat deposition in livestock and poultry were summerized.

Dairy products are an important source of high quality protein supplement in people’s lives, with high nutritional value and many health benefits. The increasing consumer demand for dairy products has brought opportunities and challenges to dairy production, and improving the productivity of dairy cows has become a hot research topic. However, modern dairy production faces many dilemmas, such as heat stress in dairy cows due to high summer temperatures, resulting in reduced fertility. Heat stress in dairy cows has adverse effects on embryo quality, including embryonic cell cycle progression and cell survival, resulting in lower embryo quality and reduced productivity. In addition, abnormal epigenetic modifications are another mechanism by which heat stress in cows affects embryo quality, mainly including abnormal histone modifications and abnormal DNA methylation. Numerous studies have shown that restoration of cellular mitochondrial function and the use of melatonin can effectively mitigate the adverse effects of heat stress on embryos. Therefore, this paper outlines the mechanisms by which heat stress leads to abnormal epigenetic modifications in embryos and thus decreases embryo quality, and two solutions to mitigate heat stress by mitochondrial transplantation and melatonin treatment to improve embryo quality, thus contributing to improve the productivity of dairy cows, with the aim of providing a reference for reducing the losses caused by heat stress in animal husbandry.

With the rapid development of animal husbandry, intensive feeding and early weaning strategy comes into being. At the same time, all kinds of stress (such as heat stress, cold stress, weaning stress, transport stress, oxidative stress) increase, resulting in intestine injury of young farm animals whose immune system has not been fully established, which is life-threatening in serious cases. miRNA is a kind of endogenous non-coding single-stranded small RNA, which can regulate gene expression. As an important member of the gene family, miRNA was involved in almost all the signaling pathways in the body. miRNA could regulate the proliferation and differentiation of intestinal epithelial cells and mediate intestinal mucosal barrier injury. This review summarized the effect of stress on intestinal mucosal barrier function, the regulatory role and possible pathways of miRNA on intestinal mucosal barrier function of young farm animals. At the same time, the mode of miRNA participating in the action of exogenous additives was reviewed to provide a theoretical basis for nutrients targeted intervention to regulate the intestinal immune function of young farm animals, and was of great significance to improve the breeding of young farm animals.

Neutrophils make up approximately 50%～70% of all mammalian leukocytes and are considered a part of the body’s first line of defense against pathogen invasion. Neutrophils play an important role in the innate immune response of the animal body, suppressing or eliminating a variety of invading pathogens through phagocytosis, degranulation and the formation of extracellular traps. Neutrophil extracellular traps (NETs) are fibrous network consist of DNA, granulins and histones that are secreted extracellularly by neutrophils after stimulated by activators, including pathogens. Initially, researchers found that NETs played an important role in fighting bacterial infections by confining bacteria to the site of infection and inhibiting or killing them. As research gradually progressed and developed, it is found that NETs not only played an antibacterial role in bacterial infections, but also had an important role in defending against fungal, parasitic and viral infections. However, pathogens have evolved various evasion strategies to inhibit the resistance of NETs. Some pathogens even utilize NETs to promote infection, making it difficult for patients to recover. This review focuses on the formation of NETs, inducing factors and roles of NETs during pathogenic infections, with a view to providing new ideas for the study of pathogenic infection mechanisms and new ways to prevent and control infections by various pathogens.

Kobuvirus (KoV) is a single-stranded, positive-sense RNA virus in a new genus of the picornavirus family and currently includes 6 species of Aichivirus A-F. KoV can infect humans and animals through direct or indirect contact. The main clinical symptom of infected animals is diarrhea. The virus is widely distributed all over the world. To date, KoV has been detected in 17 countries including the USA, China, Japan, Pakistan, Brazil, Germany, France, Tunisia, etc., suggesting that KoV has been widely distributed around the world. Recently, our studies suggested that the virus has been widely circulating among goats and sheep in China, presenting a unique evolution trend, and new genotypes have been determined. To provide a reference for KoV research, this review describes the latest research progress on KoV, including the genome structure and its coding protein, epidemiology, biological characteristics, pathogenesis and detection methods.

The relationships between animal-parasitic nematodes and their symbiotic bacteria are the key and basis for the prevention and control of these parasites. With the development of metagenomic sequencing, bioinformatics analysis, bacterial isolation and culture technology, the complex relationships between animal-parasitic nematodes and their symbiotic bacteria are gradually being unveiled. Studies showed the close relationships between parasitic nematodes and their symbiotic bacteria including nutrient interaction, immune interaction and niche effect, which promote their coexistence in same hosts. However, because of experimental purposes, experimental techniques and animal host specificity of parasitic nematodes, studies on relationships between parasitic nematodes and their symbiotic bacteria are few and scattered, and a systematic and comprehensive review is still lacking so far. Therefore, this review aims to summarize the latest research progress on relationships between animal-parasitic nematodes and their symbiotic bacteria based on current literature knowledge to increase the knowledge and understanding of this field and provide information reference for the discovery of new interventions for parasitic nematodes.

Parasitic diseases are the most destructive and pervasive infectious diseases in the world, killing tens of thousands of people annually and causing huge economic losses. Halofuginone is a halogenated derivative of febrifugine isolated and extracted from the plant Changshan, which has strong antiprotozoal activity. Compared with febrifugine, halofuginone has less toxic and side effects, which makes it more advantageous in disease treatment. In recent years, the biological activities of halofuginone in cancer, fibrosis and autoimmune diseases have attracted extensive attention. In human clinical practice, the research on the effects of halofuginone on Duchenne muscular dystrophy and solid tumors has entered the stage of clinical trials. In veterinary clinical practice, halofuginone hydrobromide and halofuginone lactate have been authorized by the FDA and the EU for the prevention and treatment of poultry coccidiosis and ruminant cryptosporidiosis, respectively. Moreover, halofuginone also has efficient inhibition on protozoa parasites such as Plasmodium, Toxoplasma, Theileria, and Leishmania. Aminoacyl-tRNA synthetases are emerging targets for the treatment of parasitic diseases, this review summarizes the effect of halofuginone on various protozoa parasites and the related mechanisms of inhibiting prolyl-tRNA synthetase, and hope to provide a reference for the subsequent theoretical research and clinical application of halofuginone antiprotozoal.

This study aimed to obtain target cells with MRC1 gene-modified PCV2 and verify the inhibitory effect of the cells on PCV2 in vitro, which would provide a theoretical basis for the subsequent production of gene-edited pigs.In this study:1) Firstly, a lentivirus-mediated high-efficiency homologous recombination vector and a CRISPR/Cas9 gene targeting system were established using genetic engineering technology; 2) The gene-edited MRC1 promoter in PK15 cells were screened by cell and molecular biology techniques; 3) Then, the anti-PCV2 effect was verified by PCV2 infection tests. The results showed that: 1) The pLV-LoxP-mCherry-puro-LoxP vector containing red fluorescent gene, puromycin gene, homologous LoxP sequence and multiple cloning sites was successfully constructed. The homologous arm was designed according to the differential sequences of MRC1 promoter among different pig breeds and the recombinant vector was constructed. Also the CRISPR/Cas9 mediated gene targeting vector were successfully constructed; 2) The PK15 cell line edited by MRC1 promoter gene was screened and verified by puromycin through virus packaging and co-transfection. The 14 bp upstream of the transcription start site of MRC1 gene was successfully knocked into PK15 cells; 3) The protein expression of MRC1 in PK15 cell line edited by MRC1 gene was significantly increased (P<0.05), and the replication of PCV2 was significantly reduced (P<0.05). The expression of MRC1 protein in PK15 cells edited by MRC1 promoter was significantly increased, which could be used for the preparation of anti-PCV2 pigs.

The study aimed to select applicable methods to assess the inbreeding degree of Huai pigs. In this study, Fhom1, Fhom2, Fvan1, Fvan2 and Fyang were calculated with “No.1 Zhongxin” porcine breeding chip data to estimate the inbreeding coefficient of 247 Huai pigs (300-days-old), and to explore the influence of SNP number on the inbreeding estimation and the correlation between different estimation methods. The results showed that the extreme variance of inbreeding coefficient Fhom1, Fhom2, Fvan1 and Fyang gradually decreased as the number of SNPs increased. When the number of SNPs reached 10 000, the inbreeding coefficients tended to be stable, which was close to the inbreeding coefficient calculated from 31 545 SNPs after quality control. The values of Fhom1, Fhom2, Fvan1, Fvan2 and Fyang were -0.216 5-0.344 0, -0.354 8-0.321 7, -0.322 8-0.892 1, -0.243 6-0.790 9, and -0.189 4-0.543 3, respectively. The values of Froh1 and Froh2 were 0.008 7-0.406 9 and 0.002 2-0.402 4, respectively. Person correlation and Spearman rank correlation of genomic inbreeding coefficients showed strong positive correlations between Fhom1 and Fhom2, Froh1, Froh5; The strong positive correlations were found between Fvan2 and Fvan1, Fyang; The strong positive correlation was observed between Froh1 and Froh5; Whereas Fhom2 was negatively correlated with Fvan1 and Fvan2. Since Froh took values from 0 to 1 that were identical to the inbreeding coefficient of a pedigree, it conformed to the evaluation habit of inbreeding coefficient; Moreover, Froh was positively correlated with the way in which genomic inbreeding coefficients were assessed, such as Fhom, Fvan and Fyang, with high applicability. The density of SNPs influenced the assessment of inbreeding coefficient, it was more appropriate to use Froh for genomic inbreeding coefficient estimation when the number and density were sufficient. The results may provide an advantageous technical means for assessing the inbreeding level of local pigs, preventing inbreeding depression and optimizing seed selection.

The purpose of this study was to explore the SNPs of FOXL2 gene and their effects on ovulation status and slaughtering traits, in order to provide theoretical basis for molecular breeding and genetic improvement of Liangguang Spotted pigs in the future. A total of 107 Liangguang Spotted pigs at the age of (365±25) days were taken as the research objects. The SNPs of FOXL2 were detected using genotyping by target sequencing (GBTS) technology, and association analysis and haplotype analysis were performed on ovulation status, teat numbers and slaughter traits. A total of 24 mutation sites were detected in the FOXL2 gene of Liangguang Spotted pigs. After eliminating SNPs with InDel mutation and the mutation frequency less than 1%, 12 SNPs were analyzed. Among them, 2 SNPs were located in the coding region and 10 SNPs were located in the non-coding region. The association analysis showed that the g.79706726 T>C, g.79706898 C>T, g.79707794 T>C, and g.79709005 T>C showed significant effects on ovulation status (P＜0.05), but had no significant effects on teat numbers trait (P >0.05). The 6 SNPs (g.79707040 A>G, g.79708079 T>C, g.79708152 G>T, g.79706726 T>C, g.79706898 C>T, and g.79707794 T>C) had significant effects on bone weight, lean meat rate, ham weight, and left carcass weight (P＜0.05), and 3 SNPs (g.79707040 A>G, g.79708079 T>C, g.79708152 G>T) displayed significant effects on leaf fat weight (P<0.05). The associa-tion analysis of haplotype combination found that the 3 SNPs g.79706726 T>C, g.79706898 C>T, and g.79707794 T>C were strongly linked (r²>0.9). The H1H2 haplotype combination (g.79706726 T>C and g.79706898 C>T) had significant effects on ovulation status, bone weight, lean meat rate, ham weight, and left carcass weight (P<0.05). The bone weight, ham weight, and left carcass traits of individuals with CCTT haplotype were significantly higher than those with TTCC haplotype (P<0.05), and the lean meat rate trait of individuals with CCTT haplotype was significantly lower than those wtih TTCC haplotype (P<0.05). Four SNPs of FOXL2 gene were significantly associated with ovulation status, which were g.79706726 T>C, g.79706898 C>T, g.79707794 T>C, and g.79709005 T>C. In addition, the SNPs of FOXL2 associated with pig slaughter traits were found in this study, indicating that FOXL2 was not only associated with ovarian phenotype and function, but also relevant for pig growth and development. This study provided an important reference for the selection and breeding of FOXL2 gene in Liangguang Spotted pigs, and also provided a theoretical basis for molecular marker-assisted breeding.

The study aimed to investigate the polymorphism of fatty acid binding protein 3 (FABP3) and stearoyl-CoA desaturase (SCD) genes and their association with meat quality traits. It can guide the breeding of Beijing black pigs at molecular level. In this study, meat quality and trait data of 413 Beijing black pigs were collected, DNA and RNA was extracted. According to FABP3 and SCD gene sequences, 20 pairs of primers were designed for PCR amplification and sequencing. DNAStar software was used to analyze the sequencing results. The correlation between FABP3 and SCD genotypes and intramuscular fat content of Beijing black pigs was analyzed and the gene expression difference was analyzed by fluorescence quantitative PCR. FABP3 gene was identified a missense mutation (c. 681 A>G), SCD gene was identified a missense mutation, two synonymous mutations and two 3'UTR region mutations. Duncan’s multiple test statistical analysis showed that all SNPs in SCD gene were not significantly associated with meat quality traits. FABP3 missense mutant c.681A>G was significantly associated with carnation L^* and intramuscular fat (IMF) content (P<0.05). The missense mutation resulted in the mutation of amino acid residue 51 of FABP3 from isoleucine (I) to threonine (T). qPCR was used to analyze the differences in individual expression levels of two homozygous genotypes of c.681A>G (AA and GG) of FABP3, and it was found that there was no significant difference in gene expression levels between the individuals with two homozygous genotypes at the mutation site. These results suggested that the missense mutation site of FABP3 c.681A>G may regulate the intramuscular fat content and carnation L^* traits by affecting the protein function. Only one locus of the FABP3 and SCD genes was significantly associated with carnation L^* and intramuscular fat content, which could be used as a candidate gene functional locus for meat quality traits of Beijing black pigs and also provide a new molecular marker for meat quality breeding of Beijing black pigs.

This study was conducted to screen out and verify differentially expressed genes and their regulatory pathways related to lipid metabolism by analyzing mRNA expression profiles in liver tissues of Wuliangshan Sooty chicken at different ages, and to provide theoretical basis for determining the expression characteristics of genes related to lipid metabolism in liver tissues of Wuliangshan Sooty chicken. In this study, D1 and D168 liver tissues were sequenced by RNA-Seq technique, and there were 3 biological replicates in each stage, transcriptome sequencing using the DNBSEQ platform, 10 differentially expressed genes were randomly selected for RT-qPCR validation. The differential genes related to lipid metabolism were screened by |log₂FC|≥2, Q value≤0.01 and KEGG pathways analysis. The differentially expressed genes of lipid metabolism were analyzed for GO enrichment, KEGG pathways, and protein interactions. A total of 100 female chicks were randomly divided into 5 groups with 20 chickens in each group. Two healthy hens were randomly slaughtered in each group at 5 stages (D1, D42, D84, D126, D168), and RNA was extracted from liver tissues. RT-qPCR was used to construct partial differential genes mRNA expression profiles. A total of 50 differentially expressed genes related to lipid metabolism were screened by transcriptome sequencing, among which 38 genes were up-regulated and 12 genes were down-regulated. The differentially expressed genes were mainly enriched in lipid metabolism, redox process, fatty acid biosynthesis, fatty acid metabolism, cholesterol biosynthesis and sterol biosynthesis. The differentially expressed genes were involved in steroid biosynthesis, fatty acid metabolism, glycerolipids metabolism, unsaturated fatty acid biosynthesis and PPAR signaling pathway. Key protein genes such as SCD, ACSBG2, SQLE, HSD17B7, LCAT and LPIN1 were screened out by protein interaction analysis, which were involved in the regulation of lipid metabolism through their mRNA expression levels. The 50 differentially expressed genes related to lipid metabolism were identified by transcriptome sequencing, and there are interactions among 42 differentially expressed genes. It provides a theoretical basis for further analysis of the molecular regulation of fat metabolism related genes in indigenous Chinese chicken.

The study aimed to examine the differentially expressed mRNAs and lncRNAs as well as their target genes in the transcriptome sequencing of Leizhou goat fetuses at 70, 90 and 120 days, and to investigate important candidate genes controlling the development of skeletal muscle in Leizhou goat fetuses, functional enrichment analysis of differentially expressed genes was carried out. Leizhou female goats were separated into 3 groups of 70, 90, and 120 days of gestation, with 3 animals in each group, for a total of 9 animals. The goats were about 2.5-3.5 years old, in the same feeding condition, healthy, and disease-free. The transcriptome was sequenced using Illumina HiSeq^TM2500 after the RNA of fetal longissimus dorsi was extracted in order to establish a library. The differential mRNAs and lncRNAs were screened at a threshold of Q<0.05, enriched for biological functions, and constructed into a target relationship network. In order to confirm the accuracy of the sequencing results, the expression levels of differential genes were lastly detected using fluorescence quantitative PCR. It was found that in the M70 vs. M90 group, a total of 185 differentially expressed mRNAs were identified, of which 170 were upregulated, 15 were downregulated, and 62 differentially expressed lncRNAs, 40 were upregulated, and 22 were downregulated. In the M120 vs. M90 group, we identified 1 048 differentially expressed mRNAs, 666 were upregulated, 382 were downregulated, 352 differentially expressed lncRNAs, 175 were upregulated, and 177 were downregulated. Among them, TNNT3, TNNI1, TNNT1, TNNI2, ITGA4, ITGA11, and ITGB4 genes were involved in regulatory pathways related to muscle development such as troponin complex, myofilament, adhesion plaque, ECM-receptor interaction, PI3K-Akt signaling pathways, etc. A total of 37 464 target genes were screened according to the positional relationship between lncRNA and mRNA for cis target gene prediction. Ten differentially expressed mRNAs were targeted by differentially expressed lncRNAs, among which NDUFB2, BRAF, METTL7B, and ITGA7 regulated adherent spots and the actin cytoskeleton, and METTL7B and ITGA7 were also engaged in PI3K-Akt signaling pathway. lncRNA TCONS_00214300 targeted METTL7B and ITGA7. Meanwhile, RT-PCR results of 8 differentially expressed mRNAs and lncRNAs showed that the expression levels of the 8 genes were similar to the expression trends of transcriptome sequencing, indicating that the sequencing results were reliable. The findings identify the lncRNAs and mRNAs that regulate fetal muscle development in Leizhou goats, as well as the potential regulatory linkages between some lncRNAs and mRNAs with differential expression. In addition, the studys also offer a theoretical foundation for molecular selection and breeding of Leizhou goats, and a novel source for studying the molecular regulatory mechanism of fetal muscle development in Leizhou goats.

The study aimed to analyze the differences, correlations and changing rules of lactating performance of Chinese Holstein cows at different lactation periods and somatic cell levels by data analysis, so as to provide a theoretical basis for the feeding and management of dairy cows at different lactation periods, the control of milk somatic cell count and the improvement of milk quality. Based on a total of 285 045 production performance measurement reports (DHI, Dairy Herd Improvement) from 14 dairy farms in Beijing, selecting healthy and normal lactating cows, 4 lactation stages were divided according to 0-99 d, 100-199 d, 200-299 d and more than 300 d (including 300 d), the low SCC group (0-20×10⁴·mL^-1), medium SCC group (20×10⁴·mL^-1-50×10⁴·mL^-1), high SCC group (more than 50×10⁴·mL^-1) were divided. A total of 251 949 DHI reports were obtained from the raw data after pretreatment, and were analyzed by Duncan multiple comparison and Pearson correlation analysis. The results showed that: 1) There were significant differences in milk yield, milk fat percentage, milk protein percentage, F/P, lactose percentage, MUN and SCC of dairy cows in different lactation periods (P<0.01). Milk yield in different lactation periods had a significant negative correlation with milk fat percentage, milk protein percentage and SCC, and a significant positive correlation with lactose percentage, and a significant positive correlation between milk protein percentage and milk fat percentage (P<0.01). 2) There were significant differences in the days of lactation, milk yield, milk fat percentage, milk protein percentage, F/P and lactose percentage of dairy cows with different levels of SCC (P<0.01). The milk yield of dairy cows at low and high levels of SCC was significantly positively correlated with SCC (P<0.01), and the medium level of SCC was significantly negatively correlated (P<0.01). The milk yield at different levels of SCC was significantly negatively correlated with milk fat percentage and milk protein percentage (P<0.01), and significantly positively correlated with lactose percentage (P<0.01). 3) The curve of milk yield changing with the lactation days was: y=-3×10^-9x⁴+3×10^-6x³-1.5×10^-3x²+0.203 9×10^-1x+34.437(R²=0.975 9); The curve of lactose percentage changing with lactation days was: y =-1×10^-15x⁶+2×10^-12x⁵-2×10^-9x⁴+6×10^-7x³-1×10^-4x²+8.4×10^-3x+4.999 2(R²=0.984 7). The variation curve of SCC with lactation days was: y=-4×10^-12x⁵+6×10^-9x⁴-3×10^-6x³+8×10^-4x²-8.89×10^-2x+22.862(R²=0.782 9); The curve of milk protein percentage changing with lactation days was: y=4×10^-15x⁶-7×10^-12x⁵+5×10^-9x⁴-2×10^-6x³+3×10^-4x²-1.94×10^-2x+3.576(R²=0.943 7); The curve of milk fat percentage changing with lactation days was: y=-6×10^-13x⁵+9×10^-10x⁴-6×10^-7x³+2×10^-4x²-1.93×10^-2x+4.777 2(R²=0.975 5); The curve of F/P changing with lactation days was: y=2×10^-11x⁴-3×10^-8x³+2×10^-5x²-2.9×10^-3x+1.44(R²=0.863 6). The milk yield and milk composition of dairy cows with different lactation stages and SCC levels showed significant differences. The milk yield and lactose percentage of dairy cows with early lactation and low SCC level were the highest. With the increase of lactation days, the milk yield and lactose percentage showed the change rule of increasing first and then decreasing; SCC, milk fat percentage and milk protein percentage showed the change trend of decreasing first and then increasing, and the F/P showed the trend of decreasing and then gradually stabilizing. Grouping and planning the dairy cows reasonably according to the lactation stage and SCC level of the dairy cows, and optimizing the nutrition supply can achieve the optimal lactation performance of the dairy cows and improve the economic benefit of the pasture.

The study aimed to study the effects of long chain non coding RNA (lncRNA) on the proliferation and differentiation of bovine skeletal muscle satellite cells. In this study, the primary skeletal muscle satellite cells of Luxi cattle fetus were isolated and cultured in vitro as experimental materials, which were induced into muscle differentiation in vitro. The specific expression lncRNA in muscle tissue screened by high-throughput sequencing in the early stage was used as the target, and the full-length amplification was carried out through RACE technology, named lncbMD, and the bioinformatics analysis and subcellular localization of lncbMD were carried out. The cells at the 1st, 2nd and 3rd day of proliferation and differentiation were collected, and RNA (4 replicates in each group) or protein (3 replicates in each group) was extracted. The expression of lncbMD in different stages of bovine skeletal muscle satellite cell differentiation was detected by qRT-PCR. Interfering RNA was designed and synthesized according to the sequence of lncbMD, and transfected into bovine skeletal muscle satellite cells to interfere with the expression of lncbMD, EdU test was used to detect the cell proliferation, qRT-PCR was used to detect the interference effect of lncbMD, the mRNA expression level of lncbMD, the mRNA expression level of proliferation markers Pax7, Cyclin D1, and differentiation markers MyoG and MyHC, and the growth of cells was observed by fluorescence inversion microscope. The results showed that the full-length sequence of lncbMD was 1 902 nt, which was located on chromosome 23 of bovine genome and had no coding potential. It was an unreported lncRNA. The expression level of mRNA in nucleus of bovine skeletal muscle satellite cells and myotubes was significantly different before and after myogenic differentiation (P<0.01). After interfering with lncbMD, the positive rate of EdU cells increased significantly (P<0.05), the mRNA expression level of proliferation marker Pax7 increased significantly compared with the control group (P<0.05), and the mRNA expression level of differentiation marker MyoG and MyHC had no significant variation compared with the control group (P>0.05). In this study, we found a new lncRNA: lncbMD, which mainly exists in the nucleus of bovine skeletal muscle satellite cells and myotubes. Further research results showed that interfering with lncbMD could promote the proliferation of bovine skeletal muscle satellite cells, but had no significant effect on the differentiation of bovine skeletal muscle satellite cells.

The study aimed to investigate the genetic diversity and evolutionary relationships of the sires from Qaidam cattle and Mongolian cattle at genomic level. In this study, the whole genome resequencing was used to scan the Y-chromosome single copy gene regions of a total of 22 individuals from 5 different geographical populations of the Qaidam cattle and 23 individuals from Mongolian cattle, and their paternal genetic diversity and phylogenetic relationship were analyzed using bioinformatics methods. The results showed that a total of 4 haplotypes were defined in 22 Qaidam cattle, with a haplotype diversity of 0.610±0.093 and a nucleotide diversity of 0.074±0.015, whereas a total of 10 haplotypes were identified in 23 Mongolian cattle, with a haplotype diversity of 0.925±0.025 and a nucleotide diversity of 0.137±0.013, indicating that Qaidam cattle had lower paternal genetic diversity compared to Mongolian cattle. The phylogenetic tree and haplotype network diagrams constructed showed that the 22 Qaidam cattle were clearly divided into two haplogroups, namely Y1 (18.2%) and Y2 (81.8%), of which Y2 was the dominant haplogroup, and the Y2 haplogroup included two sub-haplogroups, namely Y2a (18.2%) and Y2b (63.6%), of which the Y2b sub-haplogroup was dominant, indicating that the Qaidam cattle had both Y1 and Y2 paternal origins. Mongolian cattle also had both Y1 (17.4%) and Y2 (82.6%) paternal origins, but the Y2 haplogroup was dominated by the Y2a sub-haplogroup (47.8%). The above results suggest that Qaidam and Mongolian cattle have similar paternal genetic composition. However, considering the lower paternal genetic diversity of Qaidam cattle, it is suggested to increase the gene exchange of bulls from different populations within this breed for improving their genetic diversity. This study provides a theoretical basis for clarifying the paternal genetic differences between Qaidam cattle and Mongolian cattle, and for the conservation and exploitation of their germplasm resources.

The purpose of this study was to investigate the effect of fibroblast growth factor 21 (FGF21) on the apoptosis of porcine (Sus scrofa) ovarian granulosa cells and its regulatory mechanism. In this study, follicular fluid was collected from fresh ovarian tissue of Large white×Landrace sows ((8±1) months old) to separate granulosa cells as the research object. Granulosa cells were treated by RNAi technology and added with different concentrations of FGF21 recombinant protein (0, 0.1, 1, 10 ng·mL^-1) for 24 h. After 24 h, the cell apoptosis was detected by flow cytometry, and the expression levels of BAX and BCL2 in granulosa cells were investigated by Western blot, qRT-PCR. In order to further clarify the relationship between the effect of FGF21 on apoptosis of granulosa cells and mitochondria, the protein levels of mitochondrial complexes in granulosa cells after different treatments, the number of mitochondria in cells, ATP concentration, total antioxidant activity, reactive oxygen species levels and the expression levels of mitochondrial production-related genes were detected. The study results showed that after knockdown of FGF21 expression in granulosa cells by RNAi technology, the proportion of cells in late apoptosis was significantly increased (P<0.05) and the proportion of viable cells was significantly decreased (P<0.05). At the same time, the mRNA and protein levels of the pro-apoptotic gene BAX in granulosa cells in the FGF21 knockdown group were significantly increased (P<0.05), while the expression of the apoptosis-inhibiting gene BCL2 was significantly decreased (P<0.05). On the contrary, the addition of recombinant protein FGF21 effectively prevented the apoptosis of granulosa cells, and up-regulated the expression of anti-apoptotic gene BCL2 (P<0.05) and inhibited the expression of pro-apoptotic gene BAX (P<0.05). Further study found that after knockdown of FGF21 expression in granulosa cells, the expression of mitochondrial oxidative phosphorylation complex II succinate dehydrogenase complex iron sulfur subunit B (SDHB) was significantly decreased (P<0.05). At the same time, the copy number of mitochondrial DNA was significantly decreased (P<0.001); In addition, it was found that, after FGF21 knockdown, the concentration of adenosine triphosphate (ATP) and the total antioxidant capacity (T-AOC) significantly decreased (P<0.001), the expression of mitochondrial production-related genes was significantly inhibited (P<0.05), and the level of cellular reactive oxygen species (ROS) was significantly increased (P<0.05). In contrast, FGF21 recombinant protein treatment significantly increased SDHB expression (P<0.01) and mitochondrial number (P<0.05), and significantly increased mitochondrial DNA copy number (P<0.01) and ATP concentration (P<0.05), T-AOC activity (P<0.05) and expression of mitochondrial production-related genes, while ROS levels were significantly decreased (P<0.05). The above results indicated that FGF21 promoted mitochondrial biosynthesis in granulosa cells and maintained the balance of intracellular oxidation-antioxidant system, thereby inhibiting the occurrence of apoptosis of granulosa cells. This study provides a theoretical basis for further revealing the potential regulatory mechanism of follicular development in livestock.

In this study, N-acetylcysteine (NAC) was added into the culture medium of porcine oocytes for in vitro maturation to reveal the effects of NAC on the in vitro maturation of porcine oocytes from follicles with different sizes and to analyze the regulation of NAC on redox balance. Oocytes from large follicles (4-6 mm), middle follicles (2-4 mm) and small follicles (1-2 mm) in porcine ovary were collected and the three types of porcine oocytes were respectively cultured with 0, 1, 2, 4 mmol·L^-1 NAC. The maturation indexes such as cumulus expansion index and the first polar body excretion rate were evaluated, and ROS level, glutathione (GSH) content and expression level of antioxidant genes in oocytes were detected. The results showed that NAC treatment had no significant effect on cumulus expansion index and ROS level of oocytes from large follicles (P>0.05), and had no significant effect on the first polar body excretion rate of oocytes from large and middle follicles (P>0.05), however proper concentration of NAC (2 mmol·L^-1) significantly increased the cumulus expansion index and decreased ROS level (P<0.05) of oocytes from middle and small follicles (P<0.05), and significantly increased the first polar body excretion rate of oocytes from small follicles (P<0.05). Meanwhile, NAC treatment had no significant effect on GSH content and expression levels of antioxidant genes SOD, CAT, Prdx2 and Prdx6 of oocytes from large and middle follicles (P>0.05), and proper concentration of NAC (2 mmol·L^-1) significantly increased GSH content and up-regulated the expression levels of SOD and CAT of oocytes from small follicles (P<0.05). In conclusion, proper concentration of NAC (2 mmol·L^-1) can improve the in vitro maturation effect of oocytes from small follicles by alleviating oxidative stress.

The purpose of this study was to construct yak lncRNA ENSBGRT00000000387.1 overexpression lentivirus vector and infect yak follicular granulosa cells (GCs) to understand its effect on cell apoptosis. The yak follicular RNA was extracted, and the full-length sequence of lncRNA ENSBGRT00000000387.1 was amplified by RT-PCR, and then its recombinant plasmid was constructed. After it was packaged with lentivirus packaging system (pVSVG: pMDL: pRev), the infective titer on 293T cells was determined by qPCR, and then the isolated and cultured yak GCs were infected. The expression level of lncRNA ENSBGRT00000000387.1 in GCs was detected by qPCR, and the apoptosis rate of GCs was detected by flow cytometry. Western blot and qPCR were used to detect the mRNA and protein expression levels of pro-apoptotic gene CASPASE3, BAX and anti-apoptotic gene BCL-2, respectively. The full-length sequence of 2 566 bp of lncRNA ENSBGRT00000000387.1 was successfully amplified from yak follicles, and it was homologously recombined with the fragment of the homologous region of LV-EF1a-EGFP-2A vector. The restriction enzyme digestion identification and sequencing verification showed that the recombinant plasmid was successfully constructed. After it was packaged and concentrated in the lentivirus packaging system, the infection efficiency in 293T cells reached (75.77±0.850) %. The qPCR test results showed that the titer of the lentivirus was 7.32×10⁸ TU·mL^-1, indicating that the target lncRNA overexpression lentivirus vector was successfully constructed. The infection efficiency of yak follicular GCs infected with lentivirus was (52.93±1.168)%. The qPCR results showed that the expression level of target lncRNA in infected GCs was significantly increased (P<0.01). Flow cytometry showed that the apoptosis rate of cells in lentivirus infection group decreased significantly (P<0.01), the expression of pro-apoptotic genes CASPASE 3 and BAX at mRNA and protein levels decreased significantly (P<0.01), and the expression of anti-apoptotic gene BCL-2 increased significantly (P<0.01). In this experiment, lncRNA ENSBGRT00000000387.1 lentivirus vector was successfully constructed. After successfully infecting yak follicular GCs, it was found to have the effect of inhibiting apoptosis, indicating that it may play a role in yak follicular function.

This study was designed to investigate the effect of Artemisia carvifolia on anti-oxidative stress and explore its underlying mechanism in dairy cows by systematic network pharmacology. All the active ingredients of Artemisia carvifolia were obtained by the TCMSP database and the target names were converted into gene names by UniProt protein database; The keyword either “heat stress” or “oxidative stress” was used to search disease-related genes by OMIM, Gene Cards and CTD databases. The intersection of the active ingredients targets and oxidative stress targets were obtained with Venny 2.1 and then imported into the STRING database to construct a protein-protein interaction (PPI) network. Then the Cytoscape 3.9.1 software was applied for visual processing and core targets acquisition. The DAVID database performed GO and KEGG enrichment analysis on the common targets of active component and oxidative stress-related diseases. Then the Cytoscape 3.9.1 software was used again to construct the component-targets-pathway network. The results showed that, a total of 19 active components and 217 predicated targets were obtained, and 11 442 genes were targeting heat stress or oxidative stress. A total of 136 genes were intersected by components and oxidative stress. Cytoscape screened 5 key active components and 33 key targets. A total of 11 core target genes were obtained by PPI network analysis. GO function annotation showed that there are 320, 37 and 105 terms of cell components, biological processes and molecular functions, respectively, and 170 pathways were obtained by KEGG pathway enrichment analysis. Based on network pharmacological analysis, it can be concluded that the effect of Artemisia carvifolia against oxidative stress damage of dairy cows acting through multiple components, targets and pathways, thus provide a theoretical reference for further promoting the application of Artemisia carvifolia in dairy cows.

This experiment aimed to investigate the effects of feeding regimens on growth performance, slaughter performance, meat quality and the rumen bacteria community of Sunit sheep. Twenty-four healthy Sunit sheep aged 3 months with the average body weight of (20.16±1.56) kg were selected and randomly divided into 2 groups with 12 sheep in each group. The control group (confinement group) was fed a basal diet and the experimental group (pasture group) was fed natural forage. The pre-feeding period was 7 days and the trial period was 90 days. The results were shown as follows:1) The final body weight, average daily gain and chest circumference of the Sunit sheep in the confinement group were significantly higher than those in the pasture group (P<0.05). 2) The carcass weight, net meat weight, dressing percentage and meat to bone ratio were significantly higher in the confinement group than in the pasture group (P<0.05). 3)The longissimus dorsi muscle brightness (L^*) value, cooking loss and shear force of the confinement group were significantly higher than those of the pasture group (P<0.05), while the pH_{24 h} of longissimus dorsi muscle was significantly lower than that of the pasture group (P<0.05). 4) Compared to pasture feeding, the Shannon index of the confinement group was significantly decreased (P<0.05). At the phylum level, confinement feeding significantly increased the relative abundances of Bacteroidetes and Proteobacteria (P<0.05), and significantly decreased the relative abundance of Firmicutes in the rumen (P<0.05). At the genus level, confinement feeding significantly increased the relative abundance of Prevotella_1 (P<0.05), and significantly decreased the relative abundances of Rikenellaceae_RC9_gut_group and Butyrivibrio_2 in the rumen (P<0.05). Overall, confinement feeding decreases the rumen microbial diversity of Sunit sheep, improves the weight gain effect and slaughter performance of Sunit sheep, enhances cooking loss and shear force of longissimus dorsi muscle, but degrades the processing quality and eating taste of mutton.

The gut morphology and function in newborn lambs changed dramatically during suckling period. However, the growth and development of the composition and structure of gut microbiota (GM) were less clear. This experiment was conducted to study the changes and characteristics of the composition of GM in one-month-old Hu lambs. Healthy sucking lambs without antibiotic therapy history were randomly selected in neonatal period (3 days old, d03), suckling period (10 days old, d10), creep feeding period (15 days old, d15) and pre-weaning period (30 days old, d30), in addition, another 10 healthy weaned lambs at age of 60-day old (60 days old, d60) which was weaned between 43～45 days old were selected as controls, 10 lambs in each group. Rectal feces samples were collected to study the changes of the composition, structure and predicted potential function of GM by 16S rRNA. The results showed that, the number of OTU of lamb GM showed an increasing trend within one month age, but it was still extremely significantly lower than that in weaned lambs (P<0.01). The α-diversity of GM within one month age was first decreased and then increased, but it was still significantly lower than that in weaned lambs (P<0.05). The β-diversity of GM in d03 and d10 groups respectively showed an extremely significant independent distribution, but it was extremely grouped together with d15 and d30 lambs (P<0.01), and extremely independent distribution in d60 lambs (P<0.01). The relative abundance of Bacteroidetes, Spirochaetes, Lentisphaerae, Elusimicrobia, 5-7N15, CF231, Ruminococcus, Treponema and Butyricimonas in GM of one month old lambs showed an increasing trend, whereas Firmicutes, Bacteroides, Lactobacillus and Veillonella showed a decreasing trend with age growth. The carbohydrate metabolism of GM was significantly higher, while the amino acid metabolism and energy metabolism were significantly lower of lambs aged 3~15 days than that in weaned lambs (P<0.05). It is concluded that the composition, structure and predicted material metabolism function of GM in one month old lambs showed significantly dynamic changes, the richness and diversity of GM in weaned lambs are significantly increased. The changes of GM and their potential functions present a “watershed-like” characteristics of lambs aged 15~30 days, the association of this characteristic with the introduction of solid feed is worth further studying.

This experiment was conducted to investigate the effects of zearalenone (ZEA) addition with different dosages on growth performance, gastrointestinal fermentation pattern and bacterial community structure of Xiangdong black goats. Twenty-four female Xiangdong black goats with similar body weight ((12.82±2.03) kg) were randomly divided into three groups and fed with 0, 100 and 500 μg·kg^-1DM ZEA diet, respectively. The pre-feeding period lasted for one week, and the formal period was kept for four weeks. At the end of the experimental period, all the experimental animals were slaughtered, rumen and colon digesta were collected, and microbial fermentation products were detected by liquid chromatography. DNA of the rumen and colon digesta was extracted and Illumina-Nova sequencing technology was used to analyze the structure of rumen and colon microflora. Compared with the control group, the final body weight, total gain, average daily gain, and dry matter intake of goats were not affected by 100 or 500 μg·kg^-1DM ZEA addition (P>0.05). 500 μg·kg^-1DM ZEA addition decreased the molar percentage of valerate in colon digesta (P<0.05). Compared to the control group, addition of low dosage of ZEA significantly decreased the Shannon index and Simpson index (P<0.05), and addition of high dosage of ZEA significantly decreased the Simpson index and Shannon’s evenness of rumen bacteria (P<0.05). High dosage of ZEA significantly decreased the relative abundance of Firmicutes in the rumen bacteria (P=0.04). The addition of low dosage of ZEA significantly decreased the relative abundance of Actinobacteriota (P=0.04), and the addition of high dosage of ZEA significantly decreased the relative abundance of Spirochaetota in the colon bacteria(P=0.04).In conclusion, dietary 100 or 500 μg·kg^-1DM ZEA did not affect the growth performance and rumen fermentation parameters, but decreased the α diversity and evenness of rumen bacteria. The low-dose ZEA addition affected the relative abundance of Actinobacteriota which was the dominant bacteria in the colon, and the high-dose ZEA addition affected the relative abundance of Firmicutes which was the dominant bacteria in the rumen and Spirochaetota which was the dominant bacteria in the colon of goats.

The aim of this study was to comparatively analyze the differences in digestive physiology, immunity and gut microbiota in Sichuan white goose with different body weight. Normal distribution analysis was performed on the body weight of 400 Sichuan white geese at 70-day-old, and 24 high weight (HW) and 24 low weight (LW) geese were selected. The immune and digestive indexes were determined, and the gut microbiota was detected by 16S rRNA sequencing technology. The results showed that the average daily gain (ADG) of HW group was significantly higher than that of LW group (P<0.05), while there was no difference in initial body weight (P>0.05). The amylase activity, organ indexes of muscle stomach, glandular stomach and ileal, mucosal thickness of duodenum and jejunum, villus height and villus height/crypt depth of jejunum and ileum were significantly higher in HW group than those of the LW group (P<0.05), but crypt depth of ileum was significantly lower than that of LW group(P<0.05). The IgA, IgM, IgG, lysozyme content, organ indexes of thymus and bursa were significantly higher in HW group than those of the LW group (P<0.05). There was no significant difference in gut microbial α-diversity between HW and LW groups (P>0.05), while β-diversity differed significantly between the two groups (R²=0.25, P=0.02). At the phylum level, it was mainly composed of Bacteroidetes (HW: 43.50%; LW: 53.69%) and Firmicutes (HW: 39.24%; LW: 26.47%). At the genus level, seven kinds of common bacteria, including Bacteroides, Desulfovibrio and Oscillibacter etc., respectively accounted for more than 1% of the relative abundance. A total of 25 (HW: 18, LW: 7) dominant bacterial markers were significantly different between the HW and LW groups (P<0.05). The dominant bacteria in the HW group were Fusobacteriales, Clostridiales and Acholeplasmatales, while the dominant bacteria in the LW group were Bacteroidales. The results suggested that the body weight of Sichuan white geese was closely related to its digestion, immune performance and gut microbiota. Good digestion and immune function can improve the digestion and absorption of nutrients and the ability to resist diseases, as well as elevated abundance of beneficial bacteria to promote weight gain.

The aim of this study was to investigate the effects of Schizochytrium fermented product level in the diet of laying hens on production performance, egg quality, serum biochemical parameters, yolk docosahexaenoic acid (DHA) content and cecal microflora. A total of 960 healthy 40-week-old Hyland Brown hens of uniform body weight were randomly divided into 4 groups of 6 replicates each, with 40 hens in each replicate. The control group was fed the basal diet, and the experimental groups I, II, and III were fed with the diets supplemented with 0.25%, 0.5% and 1% of fermented fungus of Schizochytrium, respectively. The pre-test was 7 days and the formal test period was 30 days. The results showed that: 1) The addition of Schizochytrium fermentation to the diet had no significant effect on the egg production rate, feed-to-egg ratio and egg breakage rate (P＞0.05). 2) The addition of Schizochytrium fermentation had no significant effect on egg shape index, eggshell strength, egg white height, Hastelloy unit, yolk colour, eggshell colour and eggshell thickness (P＞0.05). 3) The analysis of the serum biochemical indicators showed that the addition of Schizochytrium fermentation product had no significant effects on total serum cholesterol, triglycerides, low-density lipoprotein, total superoxide dismutase activity and malondialdehyde (P＞0.05), but the addition level of 0.25% Schizochytrium fermentation significantly increased catalase activity (P＜0.05), 0.5% addition level significantly increased total antioxidant capacity, catalase and glutathione peroxidase activities (P<0.05), and serum glutathione peroxidase activity was significantly higher in the 1% Schizochytrium fermentation group than in the control group (P<0.05). 4) The addition of Schizochytrium fermentation to the diet of laying hens significantly increased the levels of DHA, total ω-3 and ω-6 polyunsaturated fatty acids in egg yolk (P<0.05) and significantly decreased the ω-6/ω-3 ratio (P<0.05). 5) The addition of Schizochytrium fermentation to the diet of laying hens increased the abundance of cecum microorganisms. In summary, the addition of Schizochytrium fermentation under the conditions of the current experiment could increase the antioxidant capacity of laying hens, improve the balance of intestinal flora, promote the deposition of DHA in egg yolk, and reduce ω-6/ω-3. In comprehensive consideration, 0.5% Schizochytrium fermentation addition to the diet increased the DHA content and had no adverse effects on the production performance and egg quality of the hens.

The aim of this study was to evaluate the effects of dietary urea supplementation with different levels on liver ammonia metabolism and related metabolic pathways by transcriptomic method in fattening Hu lambs. Forty-two 3-month-old fattening male Hu lambs with similar body weight of (24.3±1.7) kg were selected and randomly assigned to one of three experimental diets, which supplemented with 0 (U0 group), 10 (U10 group) and 30 (U30 group) g·kg^-1 urea on the basis of basal diets, respectively. The experiment consisted of 1 week of adaptation followed by 10 weeks of dietary treatments. At the end of the feeding trial, six animals from each group were harvested. Liver tissues of each lamb were collected to measure the concentration of ammonia-N and urea-N, and the RNA was extracted for liver transcriptome analysis. The differentially expressed genes (DEGs) were identified based on |fold change|>2 and P<0.05, and gene ontology (GO) analysis and kyoto encyclopedia of genes and genomes (KEGG) annotation were conducted. The results showed that urea supplementation increased the ammonia-N concentration in liver, and the ammonia-N concentration in U10 group was significantly higher than that in U0 group (P<0.05). Compared with U0 and U10 groups, the hepatic urea-N concentration was greatly increased in U30 group (P<0.05). Transcriptome sequencing results showed that a total of 546 DEGs were obtained by pairwise comparison among all groups. Compared with U0 group, U10 and U30 group both had 85 up-regulated DEGs, and 95 and 108 down-regulated DEGs, respectively. Compared with U10 group, 58 up-regulated DEGs and 115 down-regulated DEGs were found in U30 group. GO functional annotation results suggested that these DEGs were mainly annotated in GO terms, including amine metabolism and catabolism, regulation of immune system, and so on. Compared with U0 group, KEGG enrichment analysis showed that the energy metabolic pathway of U10 group were mainly enriched in lipid metabolism, carbohydrate metabolism, metabolism of cofactors and vitamins, xenobiotics biodegradation and metabolism, and most of its genes were significantly up-regulated. While with the urea addition to the diet further increased, the metabolic pathway of U30 group were mainly associated with the immune system, including RIG-I like receptor signaling pathway, cytosolic DNA-sensing pathway and chemokine signaling pathway. Compared with U0 group, the expression of key enzyme genes such as arginase (ARG) and glutamine synthetase (GLUL) in urea biosynthesis and glutamine synthesis pathway in liver were significantly up-regulated in the groups with urea addition (P<0.05). In conclusion, dietary urea supplementation increased the concentration of urea-N in liver by up-regulating the expression of key enzyme genes in arginine biosynthesis pathway, thus affecting liver ammonia metabolism.

In this study, we investigated the effect of dicumarol (DIC) on the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV). Firstly, the cytotoxicity of DIC on Marc-145 cells was detected by CCK-8 method and calculated the half toxicity concentration (CC₅₀). Then, the effect of DIC on the proliferation of PRRSV was detected using qPCR and Western blot. Further, DIC targeted stage of PRRSV proliferation were explored by treating the cells with different administration methods. The results showed that the CC₅₀ of DIC against Marc-145 cells was 96.11 μmol·L^-1 and DIC exhibited significant anti-PRRSV activity at a concentration of 25 μmol·L^-1; DIC inhibited PRRSV proliferation in a dose-dependent manner at different times and titers. DIC had no effect on the adsorption, entry and release of the PRRSV infection cycle but significantly inhibited the replication of PRRSV. This study confirmed that DIC exhibited significant anti-PRRSV activity in Marc-145 cells at low concentrations and mainly targeted the replication phase of PRRSV, suggesting that DIC have the potential to be developed into clinical antiviral drugs or additives, providing a theoretical basis for the development of new PRRSV antiviral drugs.

The purpose of this study was to construct a MARC-145 cell line with stable expression of eukaryotic translation initiation factor 5A (eIF5A) by means of a lentiviral vector system to provide a research tool. First, the target eIF5A sequence was synthesized and the lentivirus expression vector PLV-CMV-MCS-EF1A-PURO was constructed after double enzyme digestion. HEK293T cells were co-transfected with pLV-CMV-MCS-EF1a-Puro, the lentivirus packaging plasmid psPAX2, the envelope protein pMD2.G, and the lentivirus expressing eIF5A was packaged. The harvested lentivirus was transduced into MARC-145 cells. The MARC-145 cell line that could stable overexpress eIF5A was obtained and named as MARC-145-eIF5A cell line. MTS assay confirmed that the cell viability of the constructed cell line did not change significantly. Real-time PCR, Western blot and Indirect immune fluorescence assay were used to verify the gene and protein level of PRRSV N in the constructed cell lines and control cells, the results showed that both the gene and protein level of PRRSV N in MARC-145-eIF5A cell line were significantly increased. PRRSV propagation was significantly promoted in the MARC-145-eIF5A cell line compared with the control group. In this study, the MARC-145-eIF5A cell line which is stable over expression eIF5A protein was successfully constructed. PRRSV propagation experiment confirmed that stable expression of eIF5A could promote the PRRSV propagation in MARC-145 cells.

To screen and analyze anti-pseudorabies virus (PRV) host proteins, four differential proteins were verified by Western blot and RT-qPCR based on the results of TMT proteomics. The results were consistent with the previous results of TMT proteomics, indicating that the proteomic analysis was reliable. Eukaryotic expression vectors of four proteins were constructed, and then detected by Western blot, RT-qPCR and viral plaque assays. The results showed that all the four differential proteins could inhibit PRV replication, while the IFN-β promoter activity of MCCC1 and STRAP proteins were more significant by dual-luciferase reporter gene assay. The MCCC1 and STRAP knockdown by siRNA demonstrated that STRAP significantly promoted PRV replication. In conclusion, we found that the screened STRAP protein remarkably inhibited PRV replication, which will build the foundation for the mechanism underlying interactions between PRV-host.

The purpose of this study was to explore the effect of Salmonella virulence factor SptP on MAPK-CDK6-RB pathway and the formation of macrophage extracellular traps (METs). Firstly, compare the difference of METs levels induced by Salmonella 13312 (Salmonella Choleraesuis), Salmonella 14028 (Salmonella Typhimurium) and Salmonella 13314 (Salmonella Arizona). Then, predict the Salmonella genes involved in the regulation of METs using bioinformatics method. Finally, verify the result using ChIP and MALDI-TOF experiments. The results showed that the levels of METs induced by Salmonella 13312 and Salmonella 14028 were significantly lower than Salmonella 13314 (P<0.05), suggested that Salmonella genes may regulate some key steps of the biogenesis of METs through a certain pathway. The bioinformatics prediction of Domain—Domain interaction combined with ChIP and MALDI-TOF experiments showed that the Salmonella gene SptP participated in this process, and its host target molecules may be the downstream genes, ABL1 and CDK6, of MAPK pathway. SptP may participate in the regulation of MAPK-CDK6-RB pathway by regulating the dephosphorylation of ABL1 and CDK6, this pathway may responsible for the biogenesis of METs. The results suggest that SptP-MAPK-CDK6-RB pathway may be an important pathway for Salmonella to escape the host immune system. The discovery of this pathway provides new insights for the study of the mechanism of infection and long-term carrier of Salmonella to escape the host immune system.

Whole genomes of 91 Salmonella isolates from pig-borne food chain of Henan province were sequenced by Illumina NovaSeq 6000 platform for the understanding of genomic characteristics of Salmonella typhimurium and Salmonella serotype 4,[5],12:i:-. Serotype prediction, multi-locus sequence typing, plasmid types, virulence genes and drug resistance genes were analyzed by bioinformatics. The results showed that the prevalence of Salmonella 4,[5],12:i:- (1.58%, 50/3 173) was higher than that of Salmonella typhimurium (1.29%, 41/3 173) in pig-borne food chain of Henan Province. Sequence types(STs) of 91 Salmonella isolates included ST34 (71.42%, 65/91) and ST19 (28.57%, 26/91). Typhimurium/ST34 strains and 4,[5],12:i:-/ST34 strains were the preferred hosts of some plasmids including IncHI2A _1, IncHI2 _1 and Col440I _1. Resistant genes highly carried by Salmonella serotypes 4,[5],12:i:- were blaTEM-1B_1 (72%), tet (B)_2 (90%), sul2_3 (66%), blaOXA-1_1 (34%), etc. Compared with Salmonella typhimurium isolates, Salmonella serotype 4,[5],12:i:- isolates had a higher plasmid-carrying rate, carrying a greater variety of resistance genes and virulence genes. This study confirmed for the first time that the prevalence of Salmonella serotype 4,[5],12:i:- has exceeded that of Salmonella typhimurium in pig-borne food chain of Henan Province. To a certain extent, this may explain the predominant prevalence of the Salmonella serotype among patients in the area.

We aimed to study the effects of Brucella Type IV secretes system effector protein VceC on endoplasmic reticulum stress and gonadal hormone secretory in goat trophoblast cells, and to reveal its role in Brucella infection of host cells, which is of great significance for clarifying the mechanism of intracellular survival of Brucella and miscarriage in animals. In this experiment the eukaryotic expression vector pEGFP-C1-VceC of VceC was constructed and transfected it into GTC, then the main factors of ERS, GRP78 and CHOP, were detected by Western blot method. Then the related molecules of unfolded protein reaction (UPR) were further detected by qRT-PCR and Western blot methods. The concentration of Progesterone and Estrogen secreted by GTC were detected by ELISA. The results showed that the VceC eukaryotic expression vector pEGFP-C1-VceC was constructed and transfected it into GTC successfully. The protein expression of main factors of ERS, GRP78 were significantly increased (P<0.01) at 12 and 24 h after transfection of pEGFP-C1-VceC into GTC, while CHOP protein expression was significantly decreased (P<0.05) at 24 h after transfection. According to the qRT-PCR results, the mRNA expression levels of IRE1 and XBP-1 were significantly increased (P<0.05) at 24 h after transfection of pEGFP-C1-VceC, while PERK, ATF6 and ATF4 mRNA had little change (P>0.05). According to the Western blot results, the protein expression of IRE1 was significantly increased at 12 h after transfection of pEGFP-C1-VceC (P<0.01). In addition, the content of progesterone in cell culture supernatant of the VceC transfection group decreased significantly (P<0.01) at 12 and 24 h than those in the GTC group and pEGFP-C1 transfected GTC group, while the content of Estrogen had little change (P>0.05). The results suggest that VceC can activate the ERS of GTC by activating the IRE1 pathway of unfolded protein response. Moreover, VceC protein also changes the Progesterone/Estrogen ratio in GTC.

The purpose of this study was to evaluate the effect of ten eleven translocation 1 (TET1) on the proliferation and cytokine secretion and expression of mouse uterine natural killer (uNK) cells. The endometrium of pregnant mice was collected on gestational day 9-12, and lymphocytes were isolated. uNK cells were further sorted by streptavidin magnetic bead method for culture. The RNA interference sequence of TET1 gene was synthesized and connected to RNA interference vector GM-19167 to construct interference expression plasmid, then lentivirus was packaged and virus titer was determined. uNK cells were transfected according to the ratio of lentivirus to cell number of (10-200)∶1. After 168 h, the transfection effect was detected by fluorescence microscope. uNK cells were collected and fluorescent quantitative PCR (qPCR) method was used to verify the interference effect of TET1 gene while β- actin was used as an internal reference. Cell counting kit-8 (CCK8) was used to detect the proliferation of uNK cells. Primers of interferon-γ (IFN-γ), vascular endothelial growth factor (VEGF-C) and transforming growth factor β1 (TGF-β1) were synthesized respectively and were used to detect the expression of these three cytokines by qPCR method. The results showed that the expression of TET1 in uNK cells decreased significantly after 168 h of lentivirus infection (P<0.05), indicating that the expression of TET1 gene was significantly down regulated under RNA interference. When compared with the blank control groups, the uNK cells proliferation was not affected after TET1 interference (P>0.05) which shown by CCK8 detection results. Fluorescent qPCR results showed that after TET1 interference, the expression level of TGF-β1 was increased significantly (P<0.01), while IFN-γand VEGF-C were both decreased significantly (P<0.05). These findings revealed that interference with down-regulation of TET1 gene expression did not affect the proliferation of uNK cells, but had an effect on the transcriptional level of cytokines, thereby playing an important role in the innate immunity of animals in the uterus.

To clarify the mechanism of Met-enkephalin (MENK) on the proliferation of bovine endometrial stromal cell (BESC), the BESC were co-treated with lipopolysaccharide (LPS), opioid receptor antagonists and MENK. The opioid receptor antagonists used in this study included the nonselective opioid receptor antagonist naloxone (Nx), the δ opioid receptor antagonist ICI 154129 were the μ opioid receptor antagonist CTAP, and the groups were as follows: the blank control group, the LPS treatment group, the LPS and MENK (10^-10 mol·L^-1) co-treatment group (LM group), the LPS, MENK and opioid receptor antagonists (Nx, CTAP and ICI 154129 were all 10^-10 mol·L^-1) co-treatment group and the LPS and opioid receptor antagonist co-treatment groups. Cell viability, cell cycle, the expression of cell proliferation-related genes and the expression of Wnt/β-catenin and PI3K/AK pathway proteins were detected. The results showed that the treatment of opioid receptor antagonist alone in treatment concentration or co-treatment of LPS for 24 h had no effect on cell viability (P>0.05). Compared with the LM group, the cell numbers of G1 phase increased (P<0.05) in the co-treatment groups of LPS, MENK and Nx (LMN) and LPS, MENK and ICI 154129 (LMI), the cell numbers of S phase decreased (P<0.05) in LMN group and the co-treatment group of LPS, MENK and CTAP (LMC). Compared with LM group, the gene expressions of CCN2, TGFB1 and TGFB3 decreased (P<0.01) in the groups of LMN and LMI; the CCN2 expression decreased (P<0.01) in LMC group. Compared with LM group, there were increases in the protein expressions of cyclinD1 and GSK-3β in LMN group (P<0.05), the β-catenin and GSK-3β in LMC group (P<0.01) , and the cyclinD1 in LMC group (P<0.05) . Compared with LM group, the phosphorylation level of AKT increased (P<0.05) in LMN, LMI, and LMC groups. In conclusion, both δ opioid receptor and μ opioid receptor are involved in the proliferation of MENK on BESC.

The aim of this study was to investigate the intestinal absorption characteristics of florfenicol (FF) at different concentrations, and to explore the effect of oligopeptide transporter on intestinal absorption of florfenicol. The content of florfenicol in the perfusion samples was determined by high performance liquid chromatography (HPLC), and the in vivo circulation pass perfusion model was established to investigate the effect of different concentrations of drugs on the absorption rate of florfenicol; The effect of PepT1 on florfenicol uptake was investigated by the typical substrate of oligopeptide transporter 1 (PepT1), glycine sarcosine, and the drug absorption rate constant (K_a) and effective permeability coefficient (P_eff) of florfenicol were calculated. he results show that the accuracy and precision of the established method for determination of FF content meet the detection requirements. The K_a (μg·h^-1·cm^-2) values at low (50 μg·mL^-1), medium (75 μg·mL^-1) and high (100 μg·mL^-1) concentrations were 1.48±0.02, 1.73±0.14 and 1.90±0.07, respectively, and there were significant differences between low and medium concentration groups (P<0.05). The effective permeability coefficient P_eff (×10² cm·h^-1) was 84.12±2.40, 101.28±4.57, 110.64±5.70, respectively, with significant differences among the three groups (P<0.05). The K_a and P_eff absorbed by FF across the gut increased with increasing concentration in the concentration range used in the experiment. The P_eff (×10² cm·h^-1) value was 97.2±5.30 after the addition of high concentration (100 μg·mL^-1) glycine sarcosine (GlySar), which was significantly different from that of the non-addition group. The absorption of florfenicol decreased significantly after the addition of PepT1 typical substrate. The above experimental results suggest that the absorption mechanism of florfenicol is not only simple diffusion, and PepT1 as an active transporter participates in the trans-intestinal absorption process of florfenicol.

The purpose of this study was to establish a cat acute stress model, screen diagnostic markers, and improve the definition standard of cat acute stress. Eighteen cats were randomly divided into control group, stress group A and stress group B, with six cats in each group. Physiological indexes of each group were measured, cat stress score (CSS) was assessed, blood was collected for routine blood and blood biochemical analysis; concentration of serum inflammatory factors, hormones, 5-hydroxytryptamine (5-HT) and heat shock protein-70 (HSP-70) were detected by ELISA; serum oxidative stress indexes were detected by micro-assay method. The results showed that compared with the control group, the body temperature, respiration rate, CSS, the blood urea nitrogen (BUN) content, the creatine kinase (CK) activity, the concentration of IL-1β, TNF-α, corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol (COR), epinephrine (EPI) and 5-HT were significantly increased in stress group A (P<0.05), the red blood cell (RBC) number, the concentration of hemoglobin (HGB) and IL-10, the activity of aspartic transaminase (AST) and alanine transaminase (ALT),and the total cholesterol (TC) content were significantly decreased in stress group A (P<0.05), the heart rate, the number of white blood cells (WBC), lymphocytes (LYM) and neutrophil (NEUT), the content of glucose (GLU), total protein (TP), albumin (ALB), creatinine (CRE), triglyceride (TG) and malondialdehyde (MDA), the activity of amylase (AMY), superoxide dismutase (SOD) and catalase (CAT), and the HSP-70 concentration had no significant changes in stress group A (P>0.05). Compared with the control group, the body temperature, respiration rate, CSS, the number of WBC, NEUT and RBC, the content of GLU, BUN and TG, the activity of CK, AST, SOD and CAT, the concentration of HGB, IL-1β, TNF-α, CRH, ACTH, EPI, HSP-70 and 5-HT were significantly increased in stress group B (P<0.05), the concentration of IL-10 and COR were significantly decreased in stress group B (P<0.05), the heart rate, the LYM number, the activity of ALT and AMY, the content of TP, ALB, CRE, TC and MDA had no significant changes in stress group B (P>0.05). The results suggest that the compound stress and restraint stress designed in this study can successfully establish a cat acute stress model, and screened out CRH, ACTH and CSS which can improve the definition standard of cat acute stress.

This study was aimed to evaluate the effects of the immune regulator “polyactin A” on immune function, intestinal barrier and intestinal flora in dogs modeled for ulcerative colitis (UC). Twenty-four 10- to 11-month-old Beagle dogs were randomly divided into four groups, each group contained six dogs. The Control group and the Model group were fed the basal diet, and the Low-dose group and the High-dose group were added 1 000 and 2 000 mg·kg^-1 of polyactin A to the basal diet, respectively. The Model, Low-dose and High-dose groups were established by rectal instillation of 7% glacial acetic acid at 2 mL·kg^-1 for UC model on day 0 of the experiment, The Control group was rectal instilled with the same dose of saline and continued for be fed 7 weeks. The results showed that: 1) The dogs in the Model group, Low-dose group and High-dose group showed posterior urgency, turning back to look at the abdomen, restlessness, unformed and bloody stools, and endoscopic findings showed congested and edematous intestinal mucosa with obvious ulcers 24 h after modeling. 2) Compared with the Control group, serum IL-6 was significantly higher (P<0.05) and IL-10 was highly significantly lower (P<0.01) in the Model group, while serum IL-6 and IL-10 returned to normal levels in both the Low-dose and High-dose groups. In addition, the fecal SIgA levels were also significantly higher in the Low-dose and High-dose groups compared with the Model group (P<0.05). 3) On day 49 of the test, serum LPS levels were significantly higher in the model group compared with the Control group (P<0.05); serum LPS levels in the Low-dose group and serum DAO levels in the High-dose group were significantly lower compared with the Model group (P<0.05). 4) Compared with the Control group, the intestinal flora structure of the Model, Low-dose and High-dose groups all differed significantly (P<0.05), and the relative abundance of Lachnospiraceae in the Model group increased significantly; compared with the Model group, the difference in the flora structure of the Low-dose group was not significant (P>0.05), and the difference in the flora structure of the High-dose group was highly significant (P<0.01), with Ruminiclostridium was significantly increased in the Low-dose group (P<0.05) and Megasphaera was significantly increased in the High-dose group (P<0.05). In conclusion, the addition of polyactin A to dog food can improve the immune function, the physical barrier function and the flora structure of the intestinal tract in UC-modeled dogs, and the addition of 0.2% polyactin A to dog food is recommended.

This study aimed to explore the protective effect of aurantiamide acetate (AA) on cadmium (Cd)-induced apoptosis of osteoblasts. ROS1728 cells were used as osteoblast model. ROS1728 cells were pretreated with 5 μg·mL^-1 AA for 3 h, and then treated with 20 μg·mL^-1 CdCl₂ for 6 h. Flow cytometry was applied to detect cell apoptotic rate, mitochondrial membrane potential (MMP), and content of reactive oxygen species (ROS). Western blot was used to detect the expression of Bcl-2 and Bax. After Mito-tracker Red staining, High Content Analysis System (HCAS) was used to observe and analyze cellular mitochondrial texture index and structural changes. The results showed that compared with Cd treatment group, AA pretreatment significantly inhibited Cd-induced apoptosis rate increase Bax / Bcl-2 ratio rise, MMP decrease, mitochondrial texture structure index decline and ROS accumulation (P<0.01). The results showed that AA could inhibit Cd-induced apoptosis of ROS1728 cells by maintaining the integrity of mitochondrial structure via reducing the content of peroxide.

The purpose of this experiment was to study the biological activities of Lianhua Qingwen residue and its fermentation products, and to investigate the differences among the methods of fermentation including natural, optimised fermentation 1 (4MYL) and optimised fermentation 2 (Y4ML) in terms of the alteration of the nutrient content of the residue and the antibacterial and antiviral effects of the ethanolic and aqueous extracts. Broth dilution method was used to determine the minimum inhibitory concentration (MIC) of different extracts of drug residue before and after fermentation against three pathogenic strains of Haemophilus parasuis (HPS), Streptococcus suis type 2 (SS2) and Enterotoxic Escherichia coli (ETEC); African green monkey kidney cells (Vero), African green monkey embryonic kidney cells (MARC-145) and porcine kidney cells (PK-15) were infected with pseudorabies virus (PRV), porcine circovirus type 2 infection (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), respectively. The morphological changes of cells were observed by fluorescence microscope, and the virus nucleic acid was detected and quantified by real-time fluorescence quantitative PCR as an evaluation index of the antiviral effect of drugs in vitro. The results showed that the ethanolic and aqueous extracts of Lianhua Qingwen medicinal dregs showed inhibitory effects on HPS and SS2 strains, but had no inhibitory effect on ETEC; They all showed antiviral activity against the three test viruses, but there existed certain differences in antiviral activity. After fermentation, the ethanolic and aqueous extracts enhanced their antibacterial and antiviral activities and inhibited ETEC. It can be concluded that the extract of Lianhua Qingwen residue has different degrees of antibacterial and antiviral effects, can effectively inhibit HPS, SS2 and other common pathogenic bacteria and viruses in the process of pig breeding, and antibacterial and antiviral effect are enhanced after fermentation. The above results supported that Lianhua Qingwen residue extract has the potential to be developed into animal feed or feed additive.

This study aimed to investigate the antibacterial activity of matrine against bovine Streptococcus agalactiae in vitro, and to provide theoretical basis for the development of monomer antibacterial products of Chinese herbal medicine. In this study, the bacteriostatic activity of matrine against Streptococcus agalactis was analyzed by determining the minimum inhibitory concentration and bacteriostatic curve. In the mean time, whole blood killing test, adhesion test, biofilm staining and electron microscopic observation were used to explore the effects of matrine on the resistance of Streptococcus agalactis to organism killing, host adhesion and biofilm formation. The results showed that matrine could effectively inhibit the proliferation of Streptococcus agalactiae (ATCC13813) in vitro, and the minimum inhibitory concentration of matrine against Streptococcus agalactiae was 4 mg·mL^-1, the biofilm formation of S. agalactiae was decreased as well. Scanning transmission electron microscopy showed that the surface of the cell membrane of Streptococcus agalactiae shrank, the cell wall became smooth and thin, and the formation of capsule was significantly inhibited. Matrine could significantly inhibit the adhesion of Streptococcus agalactiae to mammary epithelial cells. Quantitative PCR showed that matrine significantly decreased the expression of virulence genes ScpB, CpsA, CylE and CAMP which related to invasion and immune evasion (P<0.05), and significantly up-regulated the expression of CpsE gene (P<0.05). In addition, matrine significantly decreased the expression of Bac, Bca, Lmb, BIBA, PI-2b and FbsB genes which related to the adhesion of virulence factors (P<0.05). These results indicate that matrine can achieve the antibacterial effect by inhibiting the proliferation of Streptococcus agalactiae (ATCC13813) in vitro, destroying the integrity thus decreasing the invasion of the strain.

This experiment was conducted to study the expression of hexokinase 2 (HK2) in canine mammary tumor (CMT) and its correlation with the prognosis of dogs. Canine mammary masses from 121 clinical samples were made into paraffin sections. After histopathological analysis and interpretation, 3 samples of malignant mammary tumors and 3 samples of normal mammary gland tissues were randomly selected for high-throughput transcriptome sequencing to screen differentially expressed genes. Quantitative real time PCR (qPCR) and immunohistochemistry were applied to detect the expression of HK2 in the overall body tissues and mammary tumors, and long-term follow-up of dogs was conducted to investigate and analyze the correlation between HK2 and the clinical characteristics and prognosis of dogs with mammary tumor. The results showed that: 1) There were 33 benign and 78 malignant mammary tumors in 121 clinical samples of canine mammary masses collected in this study; 2) Thirty-six significantly and differentially expressed genes such as HK2, MEST, GDF5 and etc, were screened out by high-throughput transcriptome sequencing. Based on the verification by qPCR and the key role played in cell metabolism, and abnormal expression in some human tumors. HK2 was designated as the research object; 3) The expression levels of HK2 mRNA and protein were low in normal canine mammary gland tissues, but significantly elevated in CMT (P<0.01); 4) The expression level of HK2 was significantly correlated with tumor tissue type, tumor size, vascular invasion, Ki-67 index and prognosis (P<0.01). HK2 is highly expressed in CMT tissues, and is significantly related to the clinical characteristics and prognosis of CMT. HK2 can be potentially regarded as a diagnostic and prognostic factor of CMT.

The study mainly focuses on the feline pruritic skin diseases, the aim was to investigate and summarize the clinical features, analyzed the clinical efficacy of common treatment options, and focused on the application of oclacitinib in feline hypersensitivity associated dermatoses, and compared the efficacy with prednisolone. We collected 138 cases who visited China Agricultural University Veterinary Teaching Hospital between January 2021 and March 2022, the basic information, clinical features, treatment options, clinical efficacy, and other information were recorded and analyzed. Among the 138 cases, the breed with the highest proportion was British short hair (27.54%), the male-to-female ratio was 1.42∶1, and the average age at consultation was 2.84 years old. Although the efficacy rate of off-label use of oclacitinib in the treatment of feline hypersensitivity associated dermatoses is slightly lower than that of prednisolone, but for effective cases, the efficacy is significant (P<0.05), and there is no significant difference in the improvement of symptoms (P>0.05). This study showed that feline pruritic skin disease has no obvious breed and gender orientation, and the onset is mostly in young age. The most common type of lesions is scratches, and the most common lesions are on the head and neck. In the etiological diagnosis of pruritic skin disease in cats, hypersensitivity associated dermatoses in cats dominates, more attention should be paid to it. In terms of treatment, the combination of drugs according to the situation is better. Based on the mechanism of oclacitinib and prednisolone, the safety of the former is higher than that of the latter, so oclacitinib can be used instead of prednisolone for pruritus control of feline hypersensitivity associated dermatoses, but the oclacitinib in cats is off-label used, so further research is needed for the safety and dosage of oclacitinib in cats.

This study was conducted to investigate the infection status of BAdV-3 by TB Green Real-time PCR on 140 nasal swab samples of sick cattle characterized by runny nose, fever, cough and dyspnea from 9 large-scale cattle farms in 6 provinces in China (Sichuan Province, Shanxi Province, Henan Province, Hebei Province, Jiangsu Province and Inner Mongolia Autonomous Region), and the positive samples were tested for the fiber shaft gene. The results showed that the BAdV-3 positive rate was 36.4% among 140 samples, and BAdV-3 was detected in all provinces except for Hebei Province, with the highest detection rates in Sichuan and Henan Provinces, reaching 82.9% (29/35) and 83.3% (10/12), respectively. Among them, the traditional types accounted for 80.4% of the total positive ratio, and the strain with the deletion of the fiber shaft region accounted for 19.6% of the total positive samples. The position and number of deletions were consistent, with 237 consecutive base pairs missing in the shaft region, and the deletion type was only detected in Sichuan and Henan Provinces, with a detection rate of 24.1% (7/29) and 30% (3/10), and a field group positive rate of 100% (2/2) and 100% (1/1), respectively. The results indicate that BAdV-3 has a wide distribution in cattle farms in China, and the current epidemic virus is mainly the traditional strain of BAdV-3. The fiber shaft gene deletion strain has a unique geographical distribution in China and is mainly prevalent in Sichuan and Henan Provinces.

The study aimed to investigate the serotypes and Apx toxin genes of Actinobacillus pleuropneumoniae clinical isolates. PCR was used to identify the serotypes and detect the Apx toxin genes of APP isolates. The results showed that 64 isolates were identified as serotypes 2, 5, 7, 8, 12, 15, 19 respectively. A total of 12 different Apx virulence factor patterns were found in 7 serotypes. The results of virulence factor analysis showed that different strains of the same serotype carried different Apx toxin genes, and the strains of different serotypes may also carry the same pattern of Apx toxin genes. A. pleuropneumoniae serotype 8 was the dominant serotype in Zhejiang Province, and serotype 19 was detected for the first time in China. There is no correlation between the Apx toxin gene and the serotype.